The objectives of this proposal are twofold: First, to identify and characterize resistant variants of the Human immunodeficiency virus, type 1 (HIV-1) as they appear in patients treated with an inhibitor of the viral protease. Second, to assess the impact these isolates might have on disease progression. The HIV-1 protease plays a crucial role in viral replication. The core particle of the virus is composed of structural and enzymatic proteins which are translated as polyprotein precursors; these precursors are cleaved by the protease during core assembly. Accurate and complete processing is essential in the formation of infectious particles and, for this reason, the protease has been intensively studied as a potential target for therapeutic intervention. Several protease inhibitors have been developed which prevent precursor processing in vitro. HIV-1 infected cells treated with these inhibitors in culture produce non-infectious particles composed of unprocessed precursors, and, recently, several inhibitors of the protease have entered clinical trials. Among the likely obstacles facing this new class of anti-retroviral agents is the development of resistance. Virus variants resistant to inhibitors of the HIV-1 reverse transcriptase are readily recovered from patients on therapy. In order to evaluate the potential for resistance to the protease inhibitors, we initiated a cell culture selection scheme to produce variants of HIV-1 which are less susceptible to the effects of one of these inhibitors. We identified several mutations in the protease coding domain which, when introduced into the purified enzyme, decreased its sensitivity by as much as 60-fold. We have also carried out an analysis of the role played by the protease in assembly of the viral core particle and demonstrated that subtle defects in protease activity result in profound deficits in virus assembly and infectively. Finally, we attempted to define the mechanism by which protease-deficient HIV-1 particles lose infectively. We found that although virus particles composed of unprocessed precursors do not synthesize viral DNA upon infection of susceptible cells, the are capable of efficiently reverse transcribing their RNA genome. This suggests that the infection of these cells is inhibited at a step before reverse transcription. Although, our preliminary results indicate that viruses encoding proteases with decreased susceptibility appear after a limited period of selection in cell culture, the role for these variants in disease progression is uncertain. We will define the range of mutations which result in protease inhibitor resistance in a group of HIV-1 infected patients enrolled in a Phase II trial of a new inhibitor. In addition, we will clarify the impact these substitutes have on HIV-1 pathogenicity. We believe that the information gained by these studies will be useful in understanding the mechanism of protease inhibitor resistance, in the rational design of new protease inhibitors, and in optimizing therapy for individual patients.